This invention is in the field of ligand receptor assays, including immunoassays, for the detection of tricyclic antidepressants and selected metabolites of tricyclic antidepressants in a fluid sample. More particularly, this invention relates to methods for the synthesis of novel tricyclic antidepressant derivatives and protein and polypeptide tricyclic antidepressant derivative conjugates and labels for use in the preparation of antibodies to tricyclic antidepressant metabolites and for use in the immunoassay process.
Tricyclic antidepressants have been used to treat endogenous depression since 1957. The therapeutic dose of tricyclic antidepressants varies from person to person and to achieve an effective dose for a patient, a simple and accurate methodology is required for the measurement of tricyclic antidepressants and their metabolites in blood and urine. In addition, doses of tricyclic antidepressants above the therapeutic amount can cause toxic side effects and death. Thus, a medical need exists for antibodies and diagnostics to rapidly detect tricyclic antidepressants and tricyclic antidepressant metabolites in order to monitor tricyclic antidepressant therapy and treat tricyclic antidepressant overdose.
The preparation of antibodies to tricyclic antidepressants and tricyclic antidepressant metabolites requires the synthesis of tricyclic antidepressant derivatives in order to covalently attach the derivative to an antigenic polypeptide or protein. In addition, the tricyclic antidepressant derivative is covalently attached to various polypeptides, proteins or labels for use in screening antibodies and in the immunoassay process. The tricyclic antidepressant derivative should mimic the structure of the tricyclic antidepressant metabolites sought to be measured. Therefore, the selection and synthesis of the types of tricyclic antidepressant derivatives for covalent attachment to proteins, polypeptides or labels are critical. In addition, the tricyclic antidepressant derivatives need to be stable and soluble in an aqueous solution.
Tricyclic antidepressant compounds and conjugates for immunization and immunoassay have been described in U.S. Pat. Nos. 4,223,013, 4,275,160, 4,307,245,4,629,691 and 4,772,697 and a European Patent Application No. 86113654.7.
The present invention is directed to novel tricyclic antidepressant derivatives which are synthesized for the covalent attachment to antigens (proteins or polypeptides) for the preparation of antibodies to tricyclic antidepressants and tricyclic antidepressant metabolites. The resulting novel antigens may be used for the production of antibodies using standard methods. Once generated, the antibodies and the novel derivatives which are covalently attached to proteins, polypeptides or labels may be used in the immunoassay process.
In accordance with the present invention and as used herein, the following terms, are defined with the following meanings, unless explicitly stated otherwise.
xe2x80x9cDrugxe2x80x9d shall mean any compound or ligand which either as a result of spontaneous chemical reaction or by enzyme catalyzed or metabolic reaction, generates an intrinsic activity when administered to a biological system. The drug may be metabolized to a derivative of the drug by a biological system. Common examples of drugs and their metabolites are tricyclic antidepressants, morphine, barbiturates, tetrahydrocannabinol, phencyclidine, amphetamines, methamphetamines, opiates, benzodiazepines, cocaine, estrone-3-glucuronide, pregnanediol-glucuronide, cotinine, lysergic acid diethylamide, methadone, propoxyphene, anabolic steroids.
xe2x80x9cDrug derivativexe2x80x9d shall mean a ligand derivative, drug, drug metabolite or a drug analogue conjugated to a linking group.
xe2x80x9cDrug metabolitexe2x80x9d shall mean a compound upstream or downstream from a drug in a biochemical or metabolic pathway, or an intermediate.
xe2x80x9cLabelxe2x80x9d shall mean a signal development element or a means capable of generating a signal, for example, a dye or an enzyme. The attachment of a drug derivative to the label can be through covalent bonds, adsorption processes, hydrophobic and/or electrostatic bonds, as in chelates and the like, or combinations of these bonds and interactions.
xe2x80x9cBinding domainxe2x80x9d shall refer to the molecular structure associated with that portion of a receptor that binds ligand. More particularly, the binding domain may refer to a polypeptide, natural or synthetic, or nucleic acid encoding such a polypeptide, whose amino acid sequence represents a specific region of a protein, said domain, either alone or in combination with other domains, exhibiting binding characteristics which are the same or similar to those of a desired ligand/receptor binding pair. Neither the specific sequences nor the specific boundaries of such domains are critical, so long as binding activity is exhibited. Likewise, used in this context, binding characteristics necessarily includes a range of affinities, avidities and specificities, and combinations thereof, so long as binding activity is exhibited.
xe2x80x9cLinking groupxe2x80x9d shall mean the xe2x80x9cchemical armxe2x80x9d between the protein, polypeptide or label and a drug or drug derivative. As one skilled in the art will recognize, to accomplish the requisite chemical structure, each of the reactants must contain the necessary reactive groups. Representative combinations of such groups are amino with carboxyl to form amide linkages, or carboxy with hydroxy to form ester linkages or amino with alkyl halides to form alkylamino linkages, or thiols with thiols to form disulfides, or thiols with maleimides or alkylhalides to form thioethers. Obviously, hydroxyl, carboxyl, amino and other functionalities, where not present may be introduced by known methods. Likewise, as those skilled in the art will recognize, a wide variety of linking groups may be employed. The structure of the linkage should be a stable covalent linkage formed to attach the drug or drug derivative to the protein, polypeptide or label. In some cases the linking group may be designed to be either hydrophilic or hydrophobic in order to enhance the desired binding characteristics of the ligand and the receptor. The covalent linkages should be stable relative to the solution conditions under which the ligand and linking group are subjected. Generally preferred linking groups will be from 1-20 carbons and 0-10 heteroatoms (NH, O, S) and may be branched or straight chain. Without limiting the foregoing, it should be obvious to one skilled in the art that only combinations of atoms which are chemically compatible comprise the linking group. For example, amide, ester, thioether, thioester, keto, hydroxyl, carboxyl, ether groups in combinations with carbon-carbon bonds are acceptable examples of chemically compatible linking groups. Other chemically compatible compounds which may comprise the linking group are set forth in this Definition section and hereby are incorporated by reference.
xe2x80x9cHydrocarbylxe2x80x9d shall refer to an organic radical comprised of carbon chains to which hydrogen and other elements are attached. The term includes alkyl, alkenyl, alkynyl and aryl groups, groups which have a mixture of saturated and unsaturated bonds, carbocyclic rings and includes combinations of such groups. It may refer to straight-chain, branched-chain cyclic structures or combinations thereof.
xe2x80x9cArylxe2x80x9d shall refer to aromatic groups which have at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted.
xe2x80x9cCarbocyclic aryl groupsxe2x80x9d shall refer to groups wherein the ring atoms on the aromatic ring are carbon atoms. Carbocyclic aryl groups include monocyclic carbocyclic aryl groups and optionally substituted naphthyl groups.
xe2x80x9cMonocyclic carbocyclic arylxe2x80x9d shall refer to optionally substituted phenyl, being preferably phenyl or phenyl substituted by one to three substituents, such being advantageously lower alkyl, hydroxy, lower alkoxy, lower alkanoyloxy, halogen, cyano, trihalomethyl, lower acylamino, lower amino or lower alkoxycarbonyl.
xe2x80x9cOptionally substituted naphthylxe2x80x9d shall refer to 1- or 2-naphthyl or 1- or 2-naphthyl preferably substituted by lower alkyl, lower alkoxy or halogen.
xe2x80x9cHeterocyclic aryl groupsxe2x80x9d shall refer to groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl, and the like, all optionally substituted.
xe2x80x9cOptionally substituted furanylxe2x80x9d shall refer to 2- or 3-furanyl or 2- or 3-furanyl preferably substituted by lower alkyl or halogen.
xe2x80x9cOptionally substituted pyridylxe2x80x9d shall refer to 2-, 3- or 4-pyridyl or 2-, 3- or 4-pyridyl preferably substituted by lower alkyl or halogen.
xe2x80x9cOptionally substituted thienylxe2x80x9d shall refer to 2- or 3-thienyl, or 2- or 3-thienyl preferably substituted by lower alkyl or halogen.
xe2x80x9cBiarylxe2x80x9d shall refer to phenyl substituted by carbocyclic aryl or heterocyclic aryl as defined herein, ortho, meta or para to the point of attachment of the phenyl ring, advantageously para; biaryl is also represented as the xe2x80x94C6H4xe2x80x94Ar substituent where Ar is aryl.
xe2x80x9cAralkylxe2x80x9d shall refer to an alkyl group substituted with an aryl group. Suitable aralkyl groups include benzyl, picolyl, and the like, and may be optionally substituted.
xe2x80x9cLowerxe2x80x9d referred to herein in connection with organic radicals or compounds respectively defines such with up to and including 7, preferably up to and including 4 and advantageously one or two carbon atoms. Such groups may be straight chain or branched.
The terms (a) xe2x80x9calkyl amino,xe2x80x9d (b) xe2x80x9carylamino,xe2x80x9d and (c) xe2x80x9caralkylamino,xe2x80x9d respectively, shall refer to the groups xe2x80x94NRRxe2x80x2 wherein respectively, (a) R is alkyl and Rxe2x80x2 is hydrogen or alkyl; (b) R is aryl and Rxe2x80x2 is hydrogen or aryl, and (c) R is aralkyl and Rxe2x80x2 is hydrogen or aralkyl.
The term xe2x80x9cacylxe2x80x9d shall refer to hydrocarbyl-COxe2x80x94 or HCOxe2x80x94.
The terms xe2x80x9cacylaminoxe2x80x9d refers to (RCONCR)xe2x80x94 and (RCO2N)xe2x80x94 respectively, wherein each R is independently hydrogen or hydrocarbyl.
The term xe2x80x9chydrocarbyloxycarbonylmethylxe2x80x9d shall refer to the group ROC(O)Oxe2x80x94 wherein R is hydrocarbyl.
The term xe2x80x9clower carboalkoxymethylxe2x80x9d or xe2x80x9clower xe2x80x9chydrocarbyloxycarbonylmethylxe2x80x9d refers to hydrocarbyl-OC(O)CH2xe2x80x94 with the hydrocarbyl group containing ten or less carbon atoms.
The term xe2x80x9ccarbonylxe2x80x9d refers to xe2x80x94C(O)xe2x80x94.
The term xe2x80x9ccarboxamidexe2x80x9d or xe2x80x9ccarboxamidoxe2x80x9d refers to xe2x80x94CONR2 wherein each R is independently hydrogen or hydrocarbyl.
The term xe2x80x9clower hydrocarbylxe2x80x9d refers to any hydrocarbyl group of ten or less carbon atoms.
The term xe2x80x9calkylxe2x80x9d refers to saturated aliphatic groups including straight-chain, branched chain and cyclic groups.
The term xe2x80x9calkenylxe2x80x9d refers to unsaturated hydrocarbyl groups which contain at least one carbon-carbon double bond and includes straight-chain, branched-chain and cyclic groups.
The term xe2x80x9calkynylxe2x80x9d refers to unsaturated hydrocarbyl groups which contain at least one carbon-carbon triple bond and includes straight-chain, branched-chain and cyclic groups.
The term xe2x80x9chydrocarbyloxycarbonylaminoxe2x80x9d refers to a urethane, hydrocarbyl-Oxe2x80x94CONRxe2x80x94 wherein R is H or hydrocarbyl and wherein each hydrocarbyl is independently selected.
The term xe2x80x9cdi(hydrocarbyloxycarbonyl)aminoxe2x80x9d refers to (hydrocarbyl-Oxe2x80x94CO)2Nxe2x80x94 wherein each hydrocarbyl is independently selected.
The term xe2x80x9chydrocarbylaminoxe2x80x9d refers to xe2x80x94NRRxe2x80x2 wherein R is hydrocarbyl and Rxe2x80x2 is independently selected hydrocarbyl or hydrogen.
The term xe2x80x9cmercaptoxe2x80x9d refers to SH or a tautomeric form.
The term xe2x80x9cmethenexe2x80x9d refers to 
The term xe2x80x9cmethylenexe2x80x9d refers to xe2x80x94CH2xe2x80x94.
The term xe2x80x9calkylenexe2x80x9d refers to a divalent straight chain or branched chain saturated aliphatic radical.
The term xe2x80x9coxyxe2x80x9d refers to xe2x80x94Oxe2x80x94 (oxygen).
The term xe2x80x9cthioxe2x80x9d refers to xe2x80x94Sxe2x80x94 (sulfur).
xe2x80x9cDisulfidexe2x80x9d refers to xe2x80x94Sxe2x80x94Sxe2x80x94.
xe2x80x9cThioesterxe2x80x9d refers to 
xe2x80x9cThioetherxe2x80x9d refers to Cxe2x80x94Sxe2x80x94C.
xe2x80x9cEsterxe2x80x9d refers to 
xe2x80x9cAnalytexe2x80x9d shall mean substance of natural or synthetic origin sought to be detected and/or measured, said substance having a specific binding partner capable of a specific interaction with said analyte.
xe2x80x9cLigandxe2x80x9d shall mean a binding partner to a ligand receptor. A substance which, if detected may be used to infer the presence of an analyte in a sample, including, without limitation, haptens, hormones, antigens, antibodies, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), metabolites of the aforementioned materials and other substances of either natural or synthetic origin which may be of diagnostic interest and have a specific binding partner therefor, i.e., the ligand receptor of a ligand-receptor assay.
xe2x80x9cReceptorxe2x80x9d shall mean a receptor capable of binding ligand, typically an antibody, or a fragment thereof, but which may be another ligand, depending on assay design.
xe2x80x9cLigand-Receptor Assayxe2x80x9d shall mean an assay for an analyte which may be detected by the formation of a complex between a ligand and a ligand receptor which is capable of a specific interaction with that ligand. Ligand-Receptor assays may be competitive or non-competitive, homogeneous or heterogeneous.
xe2x80x9cImmunogenxe2x80x9d shall mean a chemical or biochemical structure, determinant, antigen or portion thereof, which elicits an immune response, including, for example, polylysine, bovine serum albumin and keyhole limpid hemocyanin (KLH).
xe2x80x9cAntigenicxe2x80x9d shall mean a chemical or biochemical structure, determinant, antigen or portion thereof which is capable of inducing the formation of an antibody.